ANTIOXIDANTS & REDOX SIGNALING Volume 00, Number 00, 2018 ª Mary Ann Liebert, Inc. DOI: 10.1089/ars.2017.7491

FORUM REVIEW ARTICLE

Challenges and Opportunities for Small-Molecule Fluorescent Probes in Redox Biology Applications

Xiqian Jiang,1 Lingfei Wang,1 Shaina L. Carroll,1 Jianwei Chen,1 Meng C. Wang,2,3 and Jin Wang1,4,5

Abstract

Significance: The concentrations of reactive oxygen/nitrogen species (ROS/RNS) are critical to various bio- chemical processes. Small-molecule fluorescent probes have been widely used to detect and/or quantify ROS/ RNS in many redox biology studies and serve as an important complementary to protein-based sensors with unique applications. Recent Advances: New sensing reactions have emerged in probe development, allowing more selective and quantitative detection of ROS/RNS, especially in live cells. Improvements have been made in sensing reactions, fluorophores, and bioavailability of probe molecules. Critical Issues: In this review, we will not only summarize redox-related small-molecule fluorescent probes but also lay out the challenges of designing probes to help redox biologists independently evaluate the quality of reported small-molecule fluorescent probes, especially in the chemistry literature. We specifically highlight the advantages of reversibility in sensing reactions and its applications in ratiometric probe design for quantitative measurements in living cells. In addition, we compare the advantages and disadvantages of small-molecule probes and protein-based probes. Future Directions: The low physiological relevant concentrations of most ROS/RNS call for new sensing reactions with better selectivity, kinetics, and reversibility; fluorophores with high quantum yield, wide wavelength coverage, and Stokes shifts; and structural design with good aqueous solubility, membrane permeability, low protein interference, and organelle specificity. Antioxid. Redox Signal. 00, 000–000.

Keywords: fluorescent probes, sensing and imaging, hydrogen peroxide, glutathione, reversible reactions, ratiometric

- - - Introduction RNS can be categorized into anions (O2$ ,ONOO,OCl), 1 radicals ($OH, ROO$), and neutral molecules (H2O2, O2). eactive oxygen species (ROS) and reactive nitrogen Among all the ROS/RNS, the biological functions of hydrogen Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. Rspecies (RNS) are generated through intracellular me- peroxide are the most extensively studied (136). tabolism of oxygen. Oxidative damage to biomolecules, such In general, there are two classes of methods used for de- as DNA, protein, and lipids, as a result of ROS/RNS has been tecting hydrogen peroxide (H2O2) or other redox signaling linked to many pathological events (35, 48, 49, 61, 134). In molecules in living cells: genetically encoded protein probes the past decade, there has been increasing evidence that ROS/ and small-molecule-based probes. Genetically encoded pro- RNS also serve as important signaling molecules (33, 112, 130). tein probes utilize naturally occurring redox-responsive do- In cells, ROS/RNS establish redox homeostasis with antioxi- mains for sensing. Several recent review articles, including the dants, such as glutathione (GSH), cysteine (Cys), NADH, and ones in this issue, have comprehensively summarized geneti- NADPH, to regulate various biological processes, including cally encoded probes for H2O2, GSH, and thioredoxin (6, 9, 12, development and immune responses (32, 38, 130, 174). ROS/ 44, 46, 47, 105, 108, 114, 125).

Departments of 1Pharmacology and Chemical Biology, 2Molecular and Human Genetics, 4Department of Molecular and Cellular Biology, 3Huffington Center on Aging, and 5Center for Drug Discovery, Baylor College of Medicine, Houston, Texas.

1 2 JIANG ET AL.

The best known examples of reaction-based small-molecule Key Challenges for Designing probes are the calcium probes developed by Roger Tsien, such Small-Molecule Fluorescent Probes as Fura-2 and Indo-1 (150). Since the development of these Discovering sensing reactions probes, numerous fluorescence-based small-molecule probes with reaction centers have been developed, especially in the Discovering a reaction specific to the intended analyte is field of redox signaling (14, 22, 53, 68, 145, 159, 166). Due to fundamental to designing a reaction-based small-molecule the high reactivity of most of the redox signaling molecules, the probe. In the case of protein-based probes, such as redox- selectivity of the corresponding small-molecule probes still has sensitive green fluorescent protein (roGFP) or hydrogen sufficient room for improvement. Therefore, it remains a chal- peroxide sensor protein (Hyper), scientists can take advan- lenge to chemically distinguish between similar redox-active tage of existing sensing moieties from naturally occurring small molecules using small-molecule-based probes (164). protein structures (105). In contrast, the sensing reactions for As biological research has advanced, an increasing number small-molecule-based probes mostly originate from ‘‘intel- of redox signaling molecules have been uncovered (130, ligent design.’’ 136). Popular probes that were once considered selective for Taking hydrogen peroxide as an example, only a few their targeted analytes are now known to cross-react with chemical reactions have been reported to detect hydrogen newly discovered redox signaling molecules. For example, peroxide in living cells. Among the reported H2O2 probes, dichlorodihydrofluorescein (DCFH) and boronate-based the majority of them are based on the oxidation of boronate probes were initially considered to be H2O2 specific probes. esters (111, 145). Other sensing reactions for hydrogen However, recent studies revealed that they cross-react with peroxide include oxidation-induced C-C bond cleavage of other species in the redox signaling chain, such as perox- benzils (2), C-S bond cleavage of perfluoro-benzyl sulfo- ynitrite (70, 137). This will be further discussed in later nates (106), and C-N bond cleavage of anilines (82); direct sections. Therefore, it is very important for redox biologists oxidation of phosphorous (138), tellurium (78), and sele- to fully understand the sensing mechanism and limitations of nium (90, 179); and direct metal chelation by hydrogen the probe to draw appropriate conclusions. peroxide (42). Most of the reaction-based probes have been In this review, we will not only summarize redox-related extensively reviewed (14, 55, 145). Here, we discuss several small-molecule fluorescent probes but also lay out the challenges aspects of the design criteria for ideal probes, including for probe design to help redox biologists independently evaluate selectivity, kinetics, and reversibility of the sensing reac- the quality of reported small-molecule fluorescent probes. tions (Fig. 1).

FIG. 1. Desired features for small-molecule-based fluorescent probes. Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. SMALL-MOLECULE REDOX FLUORESCENT PROBES 3

Selectivity of sensing reactions In our protocol, live cells were treated with RealThiol first, then lysed in trichloroacetic acid to prevent the dis- A selective sensing reaction is the key to designing small- sociation of RealThiol from protein thiols or small- molecule fluorescent probes. It should be noted that we pre- molecule thiols under diluting conditions. GPC with fer the term ‘‘selective’’ over ‘‘specific.’’ Although specific fluorescence detection was then used to separate probes that probes, which react with nothing but the analyte of interest, had reacted with proteins from those that had reacted with are always desired, hardly any probes are truly specific due to small-molecule thiols. We found that the majority (>90%) the complexity of biological systems. Therefore, we prefer to of RealThiol had, indeed, reacted with GSH. However, this use the term ‘‘selective’’ to describe probes that preferen- method cannot be easily applied to H2O2 probe develop- tially react with the desired analyte at physiological condi- ment as the final products of the reacted probes are the same tions. It is important to fully evaluate the reactivity of the regardless of the nature of the oxidants. A possible exper- designed probe and any analytes that could potentially in- imenttotestH2O2 probe selectivity is to mix a panel of terfere under physiologically relevant conditions. physiologically relevant oxidants to determine the reaction Taking the development of H2O2 probes as an example, selectivity. If possible, cell lysate should also be added to DCFH was widely used as an H2O2 probe (23). However, the the mixture to evaluate any potential interference from complete mechanism for the oxidation of DCFH to di- intracellular proteins. chlorofluorescein (DCF) involves multiple steps and many oxidants are involved, including oxidized glutathione Kinetics of sensing reactions (GSSG), NAD, oxygen (O2), iron, and nitric oxide (NO$) (70, 71). Therefore, the fluorescent signal changes of DCFH to Ideal sensing reactions should have relatively fast kinetics DCF cannot be solely attributed to changes in H2O2 levels because the analytes are usually only present in the lM or and, indeed, reflect the overall redox state of the cells. even nM concentration range. The physiologically relevant Advancing from DCFH, several oxidation-induced C-X concentration of H2O2, for example, is 1–100 nM (136). (X = C, S, or N) bond cleavage reaction-based H2O2 probes Considering a typical probe staining concentration of 10 lM, were developed. The first reactive center used for H2O2 the reaction should have a second-order rate constant of at sensing was perfluoro-benzyl sulfonate (106, 172). Fluor- least 278 M-1$s-1 (assuming a constant 100 nM concentration ophores are linked with quenchers through this cleavable ester of H2O2) to ensure sensing results will be available within bond, enabling fluorogenic responses in the presence of H2O2. 1 h, and given the detection limit of the instrument toward the Unfortunately, the reactivity of these acyl sulfonates toward corresponding fluorophore is 1 lM. Longer incubation times other ROS was not extensively evaluated, partially due to the usually result in more artifacts, such as re-synthesis of the lack of knowledge of other redox signaling molecules at the signaling molecule being detected, metabolic inactivation of time of probe development. Perfluoro-benzyl sulfonate is probes, probe clearance from cells, and photobleaching of the prone to reductive and nucleophilic reagents from an organic fluorophores (164). chemistry perspective. Therefore, strong reductive redox sig- An ideal sensing reaction should occur in real time or at naling molecules such as hydrogen sulfide (H2S) or strong least be able to achieve a reasonable signal readout within nucleophilic molecules such as cysteine or glutathione are 5 min. Unfortunately, it is often a tradeoff between reaction highly likely to react with this reactive moiety. selectivity and kinetics. Faster reactions are usually corre- Recently, Chang and co-workers pioneered the use of lated with lower energy barriers for oxidation, meaning it boronate esters as selective reductants for H2O2, which led to may be difficult to maintain reaction selectivity, especially in a series of new H2O2 probes (4, 14, 28, 31, 34, 37, 111). The cases where the sensing reaction is irreversible and there are selectivity of this reaction has been examined against a series several molecules in the environment with similar reactivity. - of oxidants in cells, including superoxide (O2$ ), NO$, hy- Such sensing reactions have not been well established in the - droxyl radical (OH$), hypochlorite (OCl ), ozone (O3), and development of small-molecule-based H2O2 probes. Most of 1 singlet oxygen ( O2). It should be noted that peroxynitrite the H2O2 probes reported based on boronates have second- (ONOO-), which is generated intracellularly by nitric oxide order rate constants of 0.1–1.0 M-1$s-1, indicating a typical and hydroxyl radical, was later shown to react with boronates incubation time of >30 min for detecting a change in H2O2 at a much faster rate (over a million times) than H2O2 inapH concentration of 100 lM, far higher than its physiological 7.4 buffer (137). Although the intracellular concentration of concentration (110). peroxynitrite is much lower than that of H2O2, care should be The ultimate goal for redox molecule sensing is to find Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. taken when interpreting results from boronate probes. or design a fast but specific chemical reaction against the The development of GSH probes faces a similar challenge. analyte of interest (10). Another possibility is to design re- There are many free thiol-containing species in the intracellular versible sensing reactions with proper equilibrium constants milieu, including small-molecule thiols and protein thiols. to ensure sensors only respond to analytes at their physio- Glutathione, which is usually in the millimolar concentration logically relevant concentrations. A good example of the range, accounts for the majority of small-molecule thiols in implementation of this strategy is the development of gluta- eukaryotic cells. However, the concentration of free thiols in thione probes. Because glutathione is among the very few proteins can also be as high as the millimolar range, potentially nonprotein thiol species present at >1mM concentration in interfering with the readout of GSH probes. To provide a de- eukaryotic cells, it is feasible to develop a reversible sensing finitive assessment of the selectivity of GSH probes, particularly reaction that quickly and selectively responds to GSH with the intracellular selectivity, our group developed a gel perme- little interference from other free thiols. Several groups, in- ation chromatography (GPC)-based assay, which we used in the cluding our own, have reported fluorescent probes for real- study of our RealThiol probe (66). time imaging of GSH in living cells (17, 66, 101, 151). 4 JIANG ET AL.

Reversibility of sensing reactions be applied to react with all the GSH molecules to obtain a quantitative measurement. Complete scavenging of GSH in The reversibility of sensing reactions is a relatively com- cells can trigger oxidative stress, which is undesirable, es- plicated issue, especially for H2O2 and other ROS/RNS. By pecially in redox biology studies. definition, a reversible chemical reaction, depending on Second, if both reacted and unreacted probes have differ- conditions, can proceed in both the forward and reverse di- ent fluorescence responses, the concentration of the analyte rection. A chemical equilibrium is reached when forward and can be calculated based on the fluorescence ratio of the re- reverse reactions proceed at equal rates (Fig. 2). acted and unreacted probes at equilibrium conditions. This is Although irreversible reactions are commonly used in the usually referred to as ‘‘ratiometric’’ imaging. The key ad- development of reaction-based fluorescent probes (14), there vantage of ratiometric measurement is that its quantification are a few major advantages to using reversible reactions for results are independent of the absolute probe concentration probe development. First, because equilibrium is driven by in cells. Last but not least, live monitoring or longitudinal thermodynamics and can be established regardless of the tracking of the concentration of an analyte is only feasible by concentration of reactants, it is possible to measure the using reversible sensing reactions because irreversible concentration of an analyte using a probe with a much lower reaction-based probes are saturated over time. concentration, given there is an appropriate equilibrium Reversible sensing reactions have been applied to develop constant between the analyte and the probe. probes for several biologically relevant molecules, including This concept has been well demonstrated in the develop- calcium and GSH. Because of the complexity of the cellular ment of reversible reaction-based GSH probes, in which redox system, oxidation and reduction of these redox probes probes with concentrations in the micromolar or nanomolar does not always proceed directly through the same reaction range establish equilibria with GSH in the millimolar con- (Fig. 2). For example, H2O2 protein sensor Hyper transforms centration range (17, 66, 101, 151). If an irreversible to its oxidized form after reacting with H2O2. But the oxi- reaction-based GSH probe were used, an excess of the probe dized form cannot return to the corresponding reduced form (above the millimolar concentration of GSH) would have to by oxidizing H2OtoH2O2. Instead, Hyper utilizes the

FIG. 2. Two strategies for designing reversible fluores- cent probes. Route A is based on a direct chemical equilib- rium between the probe and the analyte, such as in the case of RealThiol glutathione probe. The reversibility of Route B is based on two dif- ferent chemical reactions. Usually, the reverse reaction iscatalyzedbyanenzyme, such as in the case of the Hyper hydrogen peroxide Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. probe. Grx, glutaredoxin; GSH, glutathione; H2O2, hydrogen peroxide. SMALL-MOLECULE REDOX FLUORESCENT PROBES 5

glutaredoxin (Grx)/GSH system for its reduction. As long as of other ROS/RNS that enable real-time monitoring of redox the reduction capability of the Grx/GSH system is stable, signals are still in great demand. Hyper can be used to determine H2O2 concentration inside cells at the expense of consuming hydrogen peroxide (105). Comparison between protein-based Therefore, regarding redox probes, a practical definition probes and small-molecule probes of reversible probes should be probes that can monitor both increases and decreases of an analyte in biological Two types of protein-based probes are widely used in re- systems on the minute-level time scale. Generally, the dox biology, roGFPs (39, 60) and Hyper (9)-based sensors. forward and reverse reactions should proceed at compa- roGFPs are often used for measuring GSH redox potentials, rable rates under physiological conditions. Increasing the and Hyper-based sensors are often used for measuring hy- reaction rate between H2O2 and its protein sensor reduces drogen peroxide. Their sensing reactions are based on thiol to the limit of detection that the sensor is able to achieve disulfide transformations that induce changes in fluorescent (114). However, it should be noted that blindly pursuing protein conformations and, thus, spectral properties. Both the lowest limit of detection for probe design is not ad- reactions are intrinsically reversible and can be used for vised. All probes have their own dynamic range of de- quantitative measurements. tection. Highly sensitive probes tend to have saturated There are several main advantages to protein-based geneti- readouts even at moderate concentrations of the analyte. cally encoded probes, including high selectivity and sensitivity, Therefore, it is important to match the dynamic range of amenability to subcellular targeting, and applicability to tissue- designed probes with the physiological concentration specific or whole-body imaging in animals. Despite these ad- range of the analyte. vantages, genetically encoded probes are relatively laborious to Of the small-molecule H2O2 probes, only a few probes claim work with due to the cell transfection process, and it is chal- applications in monitoring both increases and decreases in lenging to apply them in primary tissues, such as patient sam- H2O2, including thiol-disulfide-based and Te/Se, P oxidation- ples. In addition, high expression of sensor proteins may alter based probes (78, 138, 179, 181). However, the selectivity of cellular redox state due to rapid consumption of the corre- these sensing reactions has not been well characterized. As sponding analytes. Therefore, it is important to minimize the Kaur et al. pointed out, these probes would be better described expression level of sensor proteins to avoid artifacts. as reflecting ‘‘global redox changes’’ instead of H2O2 changes In contrast, small-molecule probes can be a great com- (73). To the best of our knowledge, these H2O2 probes have not plement to genetically encoded probes due to their conve- been used by redox biologists in their studies. nience, applicability in primary tissues, and minimal Several reversible redox probes targeting other ROS/RNS perturbance to the interrogated biological system (if a na- - - claim to monitor peroxynitrite (ONOO )/GSH (103), O2$ / nomolar to low micromolar probe concentration is used). GSH (99, 103, 158, 179, 181), and hypochlorous acid Since H2O2 probes are available in both protein- and small- (HClO)/H2S (158) ‘‘redox couples.’’ For example, the Han molecule-based versions, it provides a fair battleground for group reported several fluorescent probes for monitoring re- comparing these two sensing strategies. Small-molecule dox cycles between peroxynitrite and glutathione and defined H2O2 probes are mostly used to detect large H2O2 changes in peroxynitrite and glutathione as a redox couple (103, 179, cells, such as the change that occurs under strong exogenous 181). However, by definition, a redox couple is the two stimulation with bolus treatment of highly concentrated H2O2 species of a half-reaction involving oxidation or reduction, (15, 106). Although these probes provide information on for example Cu2+/Cu. It is questionable to define ONOO- and cellular responses under extreme conditions, they do not have GSH as a redox couple. sufficient sensitivity to detect basal levels of H2O2. In Han’s study, the reversibility of the ONOO-/GSH probe Protein-based probe Hyper is much better at monitoring was demonstrated by oxidizing the probe with peroxynitrite subtle concentration changes in H2O2 (9). In addition, Mor- first, then reducing the probe with addition of GSH. However, gan et al. recently reported a highly sensitive peroxiredoxin- peroxynitrite and GSH co-exist in cells. An appropriate test based probe that can detect basal levels of H2O2,inthelow of the reversibility of Han’s probe should be performed in a nanomolar to high picomolar range (114). Compared with mixture of peroxynitrite and GSH. In fact, Han’s probe could small-molecule-based H2O2 probes, their protein-based be potentially used as a reversible peroxynitrite probe. counterparts have superior sensitivity, dynamic ranges, re- Nonetheless, although the biological meaning of these action kinetics, and selectivity. More importantly, protein- designed redox couples is elusive, they could be a good based H2O2 probes are reversible and can provide quantita- Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. starting point for developing new reversible redox probes that tive measurements of H2O2 levels in living cells, whereas all oxidize and reduce through different mechanisms. small-molecule probes are based on irreversible reactions and In summary, reversible sensing reactions have several can only provide qualitative assessment of large changes of distinct advantages, although the design of them can be chal- more than 100 lM increase in H2O2 levels. Therefore, it is not lenging. GSH probes are a unique case; they have been surprising that redox biologists prefer protein-based hydro- successfully developed to reversibly react with their analyte gen peroxide probes in their studies. to achieve quantitative real-time imaging. It may not be Protein-based and small-molecule-based glutathione probes trivial to replicate the success of GSH probes to discover provide different but complementary measurements. RoGFP- similar reversible reactions for highly energetic ROS/RNS. based probes such as roGFP1/2 and Grx1-roGFP2 measure Chemists could potentially mimic protein sensors, such as GSH redox potentials, which are proportional to the value of Hyper, by designing small-molecule probes that can be oxi- [GSH]2/[GSSG] (39, 60). In contrast, state-of-the-art small- dized by ROS/RNS and reduced by endogenous enzymatic molecule-based GSH probes provide direct measurements of systems. Regardless, new reversible reactions for the sensing [GSH] (66, 101, 151). To the best of our knowledge, there are 6 JIANG ET AL.

no probes available that can quantify GSSG concentrations, in searching for GSH probes (3). Schiedel et al. introduced a either protein or small-molecule based. A possible work- diversity orientated library for screening and discovered a set around to measure [GSSG] in living cells is to apply both of bright coumarin-based fluorophores (131). Xie et al. de- roGFPs and reversible small-molecule GSH probes simulta- signed a screen and discovered several new sensing reactions neously, then calculate [GSSG] based on the values of for biothiols, which brought insights into the future design of 2 [GSH] /[GSSG] and [GSH]. sensing reactions (168). In terms of H2O2 probe design, we are Protein-based redox probes have been further developed by unaware of similar screening strategies being applied. In incorporating unnatural amino acids. The Ai group has been a general, a well-controlled screen is much more efficient than pioneer in this area and recently reported genetically encoded testing sensing reactions one at a time, especially under fluorescent probes for peroxynitrite (26) and hydrogen sulfide physiologically relevant complex conditions. (19). It should be noted that the protein-based probes reported by the Ai group are based on irreversible reactions with the cor- Choosing Appropriate Fluorophores responding analytes, meaning that it would be difficult to obtain quantitative measurements from their readouts or to follow Quantum yields of fluorophores dynamic changes in analyte concentrations. For any fluorophore, quantum yield is always among the We have summarized some of the key factors that redox most important characteristics. A high quantum yield can biologists need to consider while choosing between these two significantly reduce the amount of fluorophore that is nec- types of sensors in Table 1. In our opinion, for basic cell essary to administer to cells. In the case of detecting redox biology studies, protein-based reversible probes, if available, signaling molecules, low probe loading reduces consumption should be preferred due to their superiority in quantitative of the analyte of interest, minimizing interference to the measurements and real-time monitoring. Small-molecule biological system being interrogated. Currently, quantum fluorescent probes become useful when studying primary yields of fluorophores have to be determined experimen- tissues or when the corresponding protein-based probe is tally, which often requires laborious synthesis. unavailable. Further, we believe that probe developers from The Lavis group recently reported a general method to both small-molecule and protein camps need to learn from improve quantum yields in several of the most commonly used each other and come up with new ideas for redox sensing that fluorophores, which greatly advanced the imaging field (52, can address untapped biological questions. 54). Their elegant strategy replaced the freely rotating di-alkyl groups, which occur in many fluorophores, with a structurally rigid azetidine group, reducing rotation-related nonradiative Ideas for designing new reactions energy relaxation process to enhance quantum yields. As combinatorial chemistry and high-throughput screening Several new strategies, including aggregation induced has advanced in recent years, these cutting-edge techniques emission (AIE) and excited state intramolecular proton trans- have been applied in probe design (152, 153). Ahn et al. pio- fer (ESIPT), provide potentially useful mechanisms for alter- neered the use of a target-based combinatorial rosamine library ing quantum yields.

Table 1. Comparison Between Protein Sensors and Small-Molecule Sensors Time resolution Name Analyte Sensing reaction Selectivity of sensing Fluorophores References Representative protein sensors roGFP1/2 Redox potential Thiol-disulfide Mediated by protein 1–10 min GFP (39) [GSSG]/[GSH]2 exchange binding Grx1-roGFP2 Redox potential Grx thiol-disulfide Mediated by protein <1 min GFP (60) [GSSG]/[GSH]2 exchange binding Hyper H2O2 OxyR-RD Mediated by protein 5–10 min cpYFP (9) thiol-disulfide binding exchange

Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. Representative small-molecule sensors RealThiol GSH Reversible Michael Mediated by appropriate *1 min High quantum (66) addition chemical equilibrium yield coumarin QuicGSH GSH Reversible Mediated by appropriate <1 min Rhodamine- (151) nucleophilic chemical equilibrium Si-Rhodamine addition FRET pair QG-1 GSH Reversible Michael Mediated by appropriate <1 min Coumarin (101) addition chemical equilibrium PF1 H2O2 Irreversible boronate Mediated by selective *5 min Fluorescein (15) oxidative cleavage chemical reaction

FRET, Fo¨rster resonance energy transfer; Grx, glutaredoxin; GSH, glutathione; GSSG, oxidized glutathione; H2O2, hydrogen peroxide; Hyper, hydrogen peroxide sensor protein; OxyR-RD, regulator domain of E. Coli OxyR gene; PF1, peroxyfluor-1; roGFP, redox-sensitive green fluorescent protein. SMALL-MOLECULE REDOX FLUORESCENT PROBES 7

AIE probe molecules are nonfluorescent at low concen- FRET provides another strategy to design probes with trations, but locally high concentrations of probe molecules pseudo large Stokes shifts. Several FRET-based H2O2 probes induce and self-assemble through p-p interactions and trigger were reported to have large Stokes shifts (5, 41, 163). luminescence (63). Although sensitive detection of H2O2 has been demonstrated by using AIE probes in test tube experi- Design of ratiometric fluorescent probes ments, these probes are difficult to apply in cells (85, 86, 90, Calcium probe Fura-2, developed by Roger Tsien, was the 98, 160, 189). To achieve noticeable emission, AIE probes first widely used ratiometric probe for biomolecule sensing rely on high local concentrations of probe molecules, which (150). Ratiometric fluorescent probes show a shift in their can be toxic for cells. Some reported probes even require a excitation or emission spectra on reaction with their corre- loading concentration of up to 1 mM (189). AIE probes are sponding analytes. This feature is highly desirable in live cell also very hydrophobic due to the indispensable large conju- imaging because it provides an internal standard that allows gated building blocks of phenol rings. This can cause solu- for the correction of artifacts due to photobleaching, varia- bility and distribution problems in cells, which will be tions in laser intensity, and inhomogeneous probe concen- discussed in the following sections. tration inside cells. Figure 3 explains the chemical principle The ESIPT phenomenon was initially discovered in 3- behind the ratiometric measurements and the strategy to hydroxyflavone, whose fluorescence relies heavily on in- quantify analyte under different scenarios. At equilibrium tramolecular hydrogen bonding (148, 176). Based on the conditions, the concentration of an analyte is proportional to ESIPT mechanism, several H O probes were developed 2 2 the ratio of reacted and unreacted probe independent of the and their selectivity was evaluated in test tube experiments absolute probe concentration, given that the probe concen- (88, 94, 191). However, due to the complex environment tration is much lower than that of the analyte (Eq 1, Fig. 3A). inside cells with nearly ubiquitous hydrogen bond do- However, the quantitation equation can be complicated if nors and acceptors, ESIPT probes may lose most of their there is spectral overlap or if the concentrations of analyte excited state energies to the adjacent environment, instead and probe are similar (Eq 2, Fig. 3A) (67). When there is of emitting photons. As Zhao et al. pointed out, ESIPT no spectral overlap between reacted and unreacted probes, fluorophores prefer aprotic solvents, and ‘‘it is still a chal- the analyte concentration is proportional to the ratio R be- lenge to use ESIPT molecular probes in protic solvents tween reacted and unreacted probes (Eq 3, Fig. 3A). It should due to the severe interference of solvents to perturb the be noted that all these calculations are based on the pre- ESIPT process’’ (190). requisite that a chemical equilibrium is established between Current applications of ESIPT redox probes mostly require the analyte and the probe. Therefore, irreversible reaction- high percentages of co-solvents or surfactants, resulting in based probes are unsuitable for ratiometric-based quantita- potential artifacts and limited biological significance (94). tive measurements. Further optimization is still required for this type of probe to become practically useful in biological systems. Wavelengths of fluorescent probes Quantum dots are another set of emitters developed based on nanoparticle technology. Due to a different emission mecha- When designing fluorescent probes for biological appli- nism than small molecules, quantum dots have extremely high cations, it is important to consider appropriate excitation quantum yields and photostability, and their emission is rela- and emission wavelengths. Although some commercially tively insensitive to environmental changes. Resch-Genger et al. available plate readers have light sources with tunable extensively reviewed the applications of quantum dots versus wavelengths, almost all laser-based instruments can only small-molecule-based probes in cells in detail (126). Several accommodate very limited excitation wavelengths. Com- H2O2 probes based on quantum dots have been reported (56, 89, mercially available laser sources usually cover 405, 488, 129, 133, 185). It should be noted that depending on their sizes 514, 543, 594, and 633 nm, with some high-end products and surface charges, quantum dots may enter cells through alsoincluding355,441,458,532,561,611,647,and endocytosis, rendering them uniquely useful for studying H2O2 694 nm excitation wavelengths. Due to budget concerns, signalingintraffickingvesicles. most instruments are equipped with no more than five laser sources at the same time. The efficiency of excitation de- creases significantly when the excitation laser does not Stokes shift of fluorescent probes match the absorption maximum of a fluorophore. Stokes shift, defined as the difference between excitation Moreover, when a fluorophore has an excitation spectrum Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. maximum and fluorescence emission maximum, is another in between two laser wavelengths, it may create severe key factor in determining probe quality. Fluorophores with leaking artifacts, interfering with both channels simulta- small Stokes shifts are usually prone to self-quenching, neously. The same concerns apply to emission wavelengths, whereas those with large Stokes shifts are usually desired for although some instruments allow tunable emission bands. ratiometric or Fo¨rster resonance energy transfer (FRET)- When designing probes, it is important to use fluorophores based probes. Coumarin and cyanine fluorophores have in- with appropriate excitation and emission spectra to minimize trinsic push-pull structures, leading to potentially large Stokes potential cross-talk between different fluorescence channels. shifts. However, some of these structures have high molecular Developing a set of probes with different excitation weights or high polarity, potentially limiting the cell perme- wavelengths is advantageous due to the potential for multi- ability of the designed probes. A ‘‘prodrug’’ strategy can be plexing with other fluorescent probes or stains. For example, applied to enhance cell permeability by converting polar taking advantage of H2O2-responsive boronate chemistry, a probes into nonpolar pro-probes, which are then processed series of H2O2 probes were developed based on fluorescein, inside cells to regenerate the parent probes (66). rhodamine, and several other fluorophores (14, 163). These 8 JIANG ET AL.

FIG. 3. Equations for quantification of analyte concentration using ratiometric probes in different scenarios. (A) Equation 1 defines the relationship between the concentrations of analyte, probe, and reacted probe and the dissociation equilibrium constant K. Equation 2 is used when there is spectral overlap between the unreacted probe and the reacted probe. R is the signal ratio between reacted probe and unreacted probe. Rmin and Rmax are the R values at the corresponding 0 and saturated analyte concentrations. Equation 3 is used when there is no spectral overlap between the probe and the reacted probe. (B) The relationship between R and a hypothetical analyte concentration. In general, plotting Rversusanalyte concentration affords a sigmoidal relationship. In contrast, (R-Rmin)/(Rmax-R) is linear when plotted against the analyte concentration.

probes were multiplexed with a pH-sensitive probe to detect in the cytosol due to its preferential accumulation in hy- H2O2 under various pH conditions, as well as with an HClO drophobic environments. However, this does not necessar- probe to simultaneously detect HClO and H2O2 in live cells ily mean that the analyte concentration is higher in (36, 140). membranes than in the cytosol. Therefore, to detect hy- Near-infrared (NIR) fluorophores gained special atten- drophilic analytes, probes should have sufficient aqueous tion due to their potential in vivo applications and low solubility to avoid potential artifacts. LogP values, which phototoxicity (78, 169, 170, 181). As Wa¨ldchen et al. measure the partition of a molecule in an octanol-water pointed out, phototoxicity decreases exponentially when biphasic system, are commonly used in medicinal chemistry wavelength increases (157). NIR lasers also have excel- to evaluate the solubility of drug molecules. We recommend lent tissue penetration ability. Several cyanine-based integrating LogP measurements in the development of H2O2 probes have been shown to be useful for in vivo probes to ensure sufficient aqueous solubility. applications (72, 120, 123). Silicon-rhodamine-based Typical methods for increasing water solubility include fluorophores, developed by the Nagano group, recently adding carboxylic acid groups, hydroxyl groups, or other emerged as a new class of high quantum yield NIR fluor- hydrophilic groups to the probe core structure. However, ophores (80). H2O2 probes based on this class of fluor- these functional groups are usually very polar, making it ophore have also been reported (187). difficult for the probe to cross the hydrophobic plasma membrane. Several strategies have been developed to tackle Overcoming Cellular Barriers this problem. The most well-known method is replacing the Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. carboxylic acid groups with acetylmethoxyl (AM) esters, Solubility and membrane permeability of probes which are hydrophobic enough to cross the plasma membrane The intracellular environment is far more complicated than but cleavable by intracellular esterases so that the acid form a homogenous mixture of proteins and small molecules, but it of the parent probe is regenerated once inside the cell (69, is spatially organized. The overall concentration of proteins 147, 149). In addition, the acid form of probes is usually cell can be as high as 5 mM, but these proteins are neither ran- impermeable, which prevents the probe from leaking out of domly nor evenly distributed inside cells. Instead, there are the cells once it has been internalized. many organelles or compartments separated by lipid bilayers, The AM ester strategy has been applied in many redox creating hydrophilic pockets with hydrophobic boundaries. probes. It should be noted that the hydrolysis of AM esters Therefore, the solubility of probe molecules can bias their releases formaldehyde, which may cause toxicity and/or localization and sometimes fluorescence properties as well. alter the cellular redox state. Lowering the probe loading For example, a hydrophobic fluorescent probe tends to concentration can help to reduce the impact of the probe on show higher fluorescence readouts in lipid membranes than cells. SMALL-MOLECULE REDOX FLUORESCENT PROBES 9

Protein interference with sensing reactions measuring the half-life of probe clearance. In the event of rapid ROS/RNS are highly reactive small molecules, and their probe clearance, time-lapsed experiments with relatively long concentrations are tightly regulated in cells by a number of incubation times may not be feasible. To address this problem, redox-active enzymes. To detect ROS/RNS, probe molecules one could try to use ABC transporter inhibitors, such as pro- must react quickly enough with ROS/RNS to compete with benecid, to reduce probe clearance (155, 156). If the clearance highly efficient ROS/RNS scavenging enzymes. For exam- mechanism is unclear or the clearance rate cannot be reduced ple, it has been reported that the most popular boronate ester to the desired extent, the best approach would be to prepare a series of samples at different time points and stain each sample sensing group for H2O2 can be outcompeted kinetically by proteins, such as glutathione peroxidases (GPx) and catalase individually by using exactly the same conditions. (70). Therefore, the signal from these fluorescent probes only Designing experiments to perform quantitative detection of redox signaling molecules in live cells can be complicated represents a small portion of the intracellular H2O2 pool. In fact, most reported boronate ester-based H O probes and requires a number of controls; however, live cell exper- 2 2 iments provide much more physiologically relevant infor- can only be used to measure increases in H2O2 concentrations in the micromolar range either from bolus administration of mation than bulk measurements using lysates. external ROS or under very special biological events, such as immune responses (36). To the best of our knowledge, we are Organelle specificity of small-molecule probes unaware of anyone successfully using small-molecule fluo- rescent probes to measure physiologically relevant concen- The subcellular dynamics of redox signaling molecules trations of H2O2 in cells. have gained increasing attention in recent years. Redox In the development of glutathione probes, the highly probes targeting mitochondria (34, 41, 97, 107, 115, 162, abundant protein thiols could also react with the probes. To 171), lysosome (76, 100, 124, 187), nucleus (37, 161), en- test this possibility, our group developed a GPC-based doplasmic reticulum (7, 167), and plasma membrane (77) method with fluorescence detection to assess protein in- have all been reported. teractions with small-molecule GSH probes (67). This For example, the triphenylphosphonium (TPP) group is method could be modified and adapted to the development commonly used in probe design for mitochondrial targeting, of other redox probes. taking advantage of the highly negative mitochondrial membrane potential (Fig. 4A) (17). Probes with this group have been used to demonstrate how treatments with drugs, Probe turnover and clearance in live cells such as rotenone or antimycin A, can reduce the mitochon- Fluorescent probes are essentially xenobiotics to cells. Cells drial membrane potential. However, the reduction in fluo- actively clear these xenobiotics through various mechanisms, rescence observed during these experiments could have been including inactivation by P450 enzymes and excretion through due to the probe dissociating from the mitochondria rather ATP-binding cassette (ABC) transporters (65). Therefore, for than from an actual decrease in the analyte concentration. live cell experiments, researchers need to take the intracellular Therefore, one must be very careful when interpreting the turnover of probe molecules into consideration, preferably results of studies done with organelle-specific probes.

FIG. 4. Two strategies for achieving organelle specific- ity for small-molecule fluo- rescent probes. (A) Chemical target strategy. A chemical targeting group, such as TPP for mitochondria targeting, canbeconjugatedwiththe probe. The accumulation of the probe-TPP conjugate in mitochondria is driven by the negative mitochondrial mem-

Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. brane potential. (B) Protein tag targeting strategy. This is a two-step strategy. Protein tag- ging systems, such as Halo and SNAP, can be fused with a POI that is specifically expressed in certain organelles. Then, a conjugate between the probe and the protein tag substrate will react with the organelle- specific protein tag to achieve organelle specificity. TPP, triphenlyphosphonium; POI, protein-of-interest. 10 JIANG ET AL.

Protein tagging technologies, such as Halo-Tag (18, 102), probes of this type have been reported, and there are few, if any, SNAP-Tag (1, 50, 75, 113, 127), CLIP-Tag (50), and ACP- common properties within the group (62, 139). Other probes Tag (51, 177), can provide an alternative strategy for or- include the first H2O2-cleavable sulfonate probe (106) and the ganelle targeting. Many small-molecule organelle targeting phenol oxidation-based probe (180). The common properties of groups are synthetically difficult to produce, whereas protein hydrogen peroxide-responsive probes are summarized in Table 2. tags are synthetically convenient and expandable to essentially all subcellular targets (Fig. 4B). For example, the Halo-tag Peroxynitrite-responsive probes protein can be fused with a nuclear-specific protein or nuclear Peroxynitrite-responsive probes are categorized into three localization sequence to specifically express the Halo-tag main groups: addition-based probes, oxidation-based probes, protein in the nucleus. Small-molecule probes can then be and other probes (Table 3). One type of addition-based probe conjugated with the Halo-tag substrate to specifically react relies on the addition of the NO moiety onto a secondary acyl with the Halo-tag protein expressed in a particular organelle. amine (91, 109). Another type relies on the electrophilic Although this method does require transfection of cells, addition of the whole molecule onto a positively charged similar to genetically encoded probes, this hybrid strategy acceptor (27, 192). Both types of addition-based probes provides a nice general method for positioning small- showed good selectivity among a panel of ROS/RNS in molecule probes in cells with subcellular resolution. in vitro experiments; however, the experiments were not performed by using physiologically relevant concentrations Small-Molecule Fluorescent Probes for Redox Signaling of all ROS/RNS. Specifically, the ONOO- was tested at a To better assist readers in assessing the performance of concentration of 10 lM or higher, which is hundreds of folds redox-active fluorescent probes using the concepts of probe higher than its physiological concentration. These probes are design, here we provide a brief summary of some of the administered to cells at low micromolar concentrations and representative probes and categorize them by their sensing require 20–30 min of incubation before acquiring signals. reactions. We also excerpt available data including reaction The last type of addition-based probes has a special design selectivity, typical staining concentration, reaction kinetics, that incorporates an addition reaction with a cyclization step and wavelengths from these reports. Comprehensive sum- (142, 173). The authors claim that such a reaction design maries of more recently developed redox fluorescent probes significantly improved selectivity, especially against hydrogen can be found in these review articles (8, 14, 21, 22, 24, 55, 87, peroxide. However, because the physiologically relevant 92, 103, 111, 144, 145, 166, 183). concentration of ONOO- is significantly lower than that of - H2O2, selective detection of ONOO is still challenging in H2O2-responsive probes cells. Even using these probes, the authors observed that sig- nals generated from probes reacting with ONOO- were at most H O -responsive probes are categorized into five groups: 2 2 *200-fold higher than those generated by other ROS/RNS if boronate-based probes (the most popular option), benzil-based all of them are at a similar concentration level. Considering the probes, chalcogenides-based probes, metal-mediated probes, physiologically relevant concentration differences, at least and other probes (Table 1). 50% of their signal may come from nonspecific reactions be- As mentioned earlier, boronate-based hydrogen peroxide tween probes and other ROS/RNS inside a cell. probes have good selectivity toward H O at micromolar 2 2 Oxidation-based probes can be further divided into three concentration levels (15, 25, 86, 98, 100, 116, 171, 189). The groups, phenol-based (88, 122), boronate-based (141, 182), cross-reactivity is typically tested in a test tube experiment and Te-based (181) probes. As mentioned earlier, most against a panel of individual ROS/RNS. The typical staining phenol-based and Te-based probes are best used as indicators concentration used for cell experiments is in the low micro- of the overall redox state. However, some in vitro experi- molar range, with the exception of the AIE-based probe that ments reveal excellent selectivity between ONOO- and other requires a staining concentration as high as 1 mM (189). To ROS/RNS, with a difference in response of up to several detect an increase of H O of *100 lM, the typical incubation 2 2 thousand folds. This is potentially due to a kinetic difference time for these probes is 30 min. Peroxyfluor-1 (PF1) only re- (122). Boronate-based probes react with ONOO- quickly, but quires a 5-min incubation due to its fast reaction with H O 2 2 due to the low concentration of ONOO- inside cells, they still (15). For benzil-based probes, the reaction kinetics are gen- require a 30-min incubation time before optimal signals can erally faster than for boronate-based probes. An incubation be detected (84, 128, 141, 182). There are also a few other time of only 10 min is needed before reading the signal (1, 2). - Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. interesting mechanisms that have been applied to ONOO Testing of cross-reactivity between benzil-based probes sensing (13). The available common properties of and other ROS/RNS is performed in the same manner as that peroxynitrite-responsive probes are summarized in Table 3. of boronate-based probes, and the results are similar. Chalcogenide-based probes take advantage of the affinity HClO-responsive probes between oxygen and chalcogenide atoms, as well as the re- versibility of such oxidation reactions. However, the selec- HClO-responsive probes are all oxidation-based probes, tivity profile for this kind of probe is not well established. To and they are categorized into three sub-types (Table 4): date, these probes seem to be more suitable as overall redox chalcogenides-based (104, 154, 184), phenol/aniline-based state probes instead of H2O2-selective probes. More detailed (64, 81, 193), and thiol ether-based probes (11, 79). These data from biological studies are required before we can fully oxidation-based probes, regardless of reaction reversibility, are assess their performance (74, 78). mostly overall redox state indicators. The selectivity tests for Metal-mediated probes mainly rely on transition metals and these probes are usually performed by comparing probe re- their affinity toward oxygen under specific conditions. Few sponses with micromolar concentrations of all ROS/RNS Table 2. H2O2-Responsive Probes Reported staining Time needed concentration before optimal lex /lem Name Species tested for cross-reactivity for cells signal available (nm) References Boronate-based probes

- PAM-BN-PB CH3COOH, TBHP, ClO , 10 lM 30–60 min 410/480 (25) O2$-, $OH, -OtBu, GSH, - Vc, ONOO ,H2O2 HP-L1 K+,Na+,Ca2+,Mg2+,Zn+, 5 lM 30 min 520/584 (100) 2+ 2+ Cu ,Fe , GSH, Cys, HClO, H2O2 $- - Mito-H2O2 HClO, O2 , OtBu, $OH, 10 lM 30–90 min 490/527 (171) TBHP, NO, ONOO-, GSH, - Vc, Glu, HSO3 ,H2O2 - $- QCy-BA OCl ,O2 , TBHP, $OH, 5 lM 30 min 400/565–680 (116) - - OtBu, NO, ONOO ,H2O2 - $- PF1 OCl ,O2 , TBHP, $OH, 5 lM 5 min 450/460–700 (15) - OtBu, NO, H2O2 - $- TPE-DABA (AIE) OCl ,O2 , TBHP, $OH, 50 lM N/A 430/576 (98) - - - OtBu, NO, NO3 , ONOO ,H2O2 TPE-BO (AIE) 1O2, NO, ClO-, TBHP, $OH, 5 lM to 1.0 mM N/A 400/500 (189) $- O2 , GSH, H2O2 D-BBO (AIE) 1O2, NO, ClO-, TBHP, $OH, N/A N/A 405/510 (86) $- O2 ,H2O2 Benzil-based probes

NBzF-BG N/A 2 lM 10 min 505/525 (1) NBzF 1O2, ONOO-, NO, ClO-, 5 lM 10 min 495/519 (2) $- TBHP, $OH, O2 ,H2O2 Chalcogenides-based probes

- - $- 2-Me TeR ONOO , NO, ClO , $OH, O2 ,H2O2 5 lM N/A (short 660/690 (78) lived) Compound 3 N/A N/A N/A 500/565 (74) Metal-mediated redox probes

- $- - ZP1Fe2 CAN, DDQ, ClO ,O2 , OtBu, 10 lM 30 min 507/533 (139) $OH, TBHP, NO, H2O2 Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. MBFh1 N/A 5 lM 1 min 570/590 (62) Other probes 1a ONOO-, NO, TBHP, ClO-, $OH, 25 lM 60 min 485/530 (106) $- $- $- O2 ,O2 +catayse, O2 +SOD, H2O2

DA-Cy $OH, MeLOOH, CuOOH, tBuOOH, 5 lM 3 min 630/755 (180) - 1 O2 , O2, ClO, H2O2

- - AIE, aggregation induced emission; N/A, not available; NO, nitric oxide; O2$ , superoxide; OCl , hypochlorite; OH$, hydroxyl radical; ONOO-, peroxynitrite.

11 Table 3. Peroxynitrite-Responsive Probes Reported Staining Time Needed Concentration before Optimal Name Species Tested for Cross-Reactivity for Cells Signal Available lex /lem (nm) References Addition-based probes

- $- - A2 OCl ,O2 , $OH, ROO , NO, 2 lM 20 min 485/517 (109) - - 1 ONOO ,H2O2,NO2 , O2 - $- - Ds-DAB OCl ,O2 , $OH, NO, NO3 , 10 lM 30 min 350/505 (91) - - ONOO ,H2O2,NO2 , HNO

- $- - MITO-CC OCl ,O2 , $OH, NO, NO3 , 5 lM 20 min 420/651 (27) - - ONOO ,H2O2,NO2 , TBHP, 2- BuO$,H2S, H2S2,SO3 , HNO - $- - CHCN OCl ,O2 , $OH, ROO , NO, 5 lM 30 min 515/635 (192) - ONOO ,H2O2, Cys, Hcy, GSH, TBHP

- $- - HKGreen-1 OCl ,O2 , $OH, ROO , NO, 20 lM 15 min 490/521 (173) - 1 ONOO ,H2O2, O2 - $- - HKGreen-2 OCl ,O2 , $OH, ROO , NO, 20 lM 60 min 520/539 (142) - 1 - ONOO ,H2O2, O2, NO, NO3 Oxidation-based probes

- $- - HKGreen-4 OCl ,O2 , $OH, ROO , NO, 10 lM 60 min 517/535 (122) - 1 ONOO ,H2O2, O2 - $- - NP3 (ESIPT) OCl ,O2 , $OH, ROO , NO, 5 lM 30 min 405/420–480 (88) - 1 ONOO ,H2O2, O2, Ala, Gly, Cys, GSH, Cu2+,Fe2+,Fe3+,Zn2+

- $- - 1-D-fructose OCl ,O2 , $OH, ROO , NO, 5 lM 30 min 410/525 (141) Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. - - - NO3 , ONOO ,H2O2,NO2 , - $- - PyBor OCl ,O2 , $OH, NO, ONOO , 10 lM 30 min 347/410 (182) H2O2, TBHP, CuOOH, BrO Fl-B N/A 50 lM 30 min 492/515 (128)

- $- - Cy-NTe OCl ,O2 , $OH, NO, ONOO , 1 lM 5 min 793/820 (181) H2O2, TBHP, CuOOH, MeLOOH

(continued)

12 SMALL-MOLECULE REDOX FLUORESCENT PROBES 13

Table 3. (Continued) Reported Staining Time Needed Concentration before Optimal Name Species Tested for Cross-Reactivity for Cells Signal Available lex /lem (nm) References Other probes

5-Fluoroisatin Detected by 19F NMR (13)

PN600 Three-channel fluorescent probe (188)

1 Cys, cysteine; ESIPT, excited state intramolecular proton transfer; O2, singlet oxygen.

including HClO, which do not represent their corresponding (Table 5). The reversible probes are based on either Michael physiologically relevant concentrations. The staining concen- addition reactions or nucleophilic addition to rhodamines; tration is usually in the low micromolar range, and the typical whereas irreversible probes use several different reactions, incubation time is 15–30 min. The available common proper- including addition and substitution reactions. ties of HClO-responsive probes are summarized in Table 4. A reversible Michael addition with a proper equilibrium constant can be applied to quantitative monitoring of gluta- thione concentrations in live cells. Since our group first in- Thiol-responsive probes troduced the concept by designing ThiolQuant-Green 2 years Thiol-responsive probes are categorized into two main ago (67), several groups including our own have followed up types: reversible and irreversible reaction-based probes and designed probes with improved reaction kinetics (16, 66,

Table 4. HClO-Responsive Probes Reported staining Time needed concentration before optimal Name Species tested for cross-reactivity for cells signal available lex /lem (nm) References

- $- - Mito-TP OCl ,O2 , $OH, NO, ONOO ,H2O2, 10 lM 20 min 375/500 (184) TBHP, BuO$,NO$, tBuOO$, ATP, ADP, NAD, GSH, Cys, Fe3+,Zn2+,Cu2+, - $- - NI-Se OCl ,O2 , $OH, NO, ONOO , 10 lM 5 min 488/490–590 (104) 1 H2O2, TBHP, O2, CHP - $- - - HCTe OCl ,O2 , $OH, NO, NO3 , ONOO , 10 lM 30 min 480/531 (154) - 1 H2O2,NO2 , TBHP, O2 Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only.

- $- BClO OCl ,O2 , $OH, NO, H2O2, 1 lM 20 min 480/505 (193) 1 TBHP, tBuO$, O2 - $- HKOCl-2 OCl ,O2 , $OH, NO, H2O2, 10 lM 30 min 523/545 (64) - 1 TBHP, ONOO , ROO$, O2 - $- - MitoAR OCl ,O2 , $OH, $NO, H2O2, ONOO 1 lM 15 min 543/560 (81)

- - Hypo-SiF OCl ,KO2, $OH, $NO, H2O2, ONOO N/A N/A 570/606 (11) - $- - MMSiR OCl ,KO2,O2 , $NO, H2O2, ONOO 1 lM 4 min 620/670 (79) 14 JIANG ET AL.

Table 5. Thiol-Responsive Probes Reported staining Time needed concentration before optimal Name Species tested for cross-reactivity for cells signal available lex /lem (nm) References Reversible reaction-based probes

RealThiol GSH, Gly, Lysate, Cys, H2S, 0.1–1 lM 1–5 min 405/487 (66) Lipoic acid, KSCN, Na2S2O3, 488/565 - NO, H2O2, HOCl, O2 , NaNO2, Protein thiols TQ-Green GSH, Cys, BSA, Lysate 1 lM 15–30 min 405/488 (67) 488/565 QG-1 H2O2, GSH 10 lM 1 min 405/488 (101) 488/560

QG3.0 H2O2, GSH 1 lM 1–5 min 558/593 (151) 609/632 Irreversible reaction-based probes

Probe 1 Glu, Ala, Arg, Val, Ser, Lys, N/A N/A 444/496 (93) Leu, Adenine, Guanine, Cytosine, Thymine, Glucose, Ca2+,Mg2+,K+,Na+,Zn2+, 3+ Fe , NADH, H2O2 1Ca2+,Mg2+,K+,Na+,Zn2+, 1 lM 5 min 400/420–600 (175) Fe3+,Cu2+,Hg2+, GSH

Probe DTT, 3¢-thio dT, Glu, Ala, 25 lM 15 min 490/522 (135) Arg, Lys, ME, Vc, H2O2

Aldehyde 1 Glu, Ala, Arg, Ser, Lys, Leu, N/A N/A 315/360 (143)

Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. Pro, His, Gln, GSH, Cys

101, 151). All these GSH probes have reaction kinetics in the Despite using similar Michael addition reactions, some seconds to minutes range, and they have been demonstrated GSH probes with suboptimal equilibrium constants and/or to quantitatively follow GSH dynamics in living cells. sluggish reaction kinetics cannot be used for time-lapsed It should be noted that these probes are claimed to be selec- experiments (93, 175). When coupling two addition reac- tive for GSH based on the fact that GSH is the most abundant tions for cyclization, several folds of selectivity between small-molecule thiol in eukaryotic cells. However, some or- different biothiols have been achieved. However, since ganisms use trypanothione (45), mycothiol (118), or bacillithiol GSH is the dominant free thiol in cells, it is difficult to (119) instead of glutathione. In these cases, the GSH probes achieve selective signal detection under physiological could be repurposed to quantify these other small-molecule conditions (143). The nucleophilicity of thiols has also been thiols assuming their concentrations are in the mM range. utilized to design substitution reaction-based thiol probes Table 6. Probes for Other Redox Metabolites Reported staining Time needed Species tested for concentration before optimal Name cross-reactivity for cells signal available lex /lem (nm) References Sulfenic acid probes

Used for labeling in cell lysates DAz-1 N/A 10 mM 2 h N/A (132) DAz-2 N/A 5 mM 2 h N/A (83)

NBD-Cl N/A 1 lM 30 min Various (43) (absorbance at 340 nm) Sulfane sulfur probe

SSP2 Cys, GSH, Hcy, GSSG, Na2S, 50 lM 20 min 482/518 (20) Na2S2O3, Na2SO3,Na2SO4, formaldehyde, acetaldehyde, Na2S2, Cys-poly-sulfide, S8 DSP-3 GSH, Cys, Hcy, GSSG, Na2S, 10 lM 20 min 490/515 (95) Na2S2O3,Na2SO3,Na2SO4, CH3SSSCH3,S8,Na2S2 Sulfite and bisulfite probes

- - - - 2- 2- Probe 1 F ,Cl,Br,I,SO4 , HPO4 . N/A N/A 487/588 (30) - - - - - NO3 ,N3 , AcO , ClO4 , HCO3

- - - - 2- P-1 AcO ,NO2 ,NO3 ,F,CO3 , 50 lM 30 min 330/395–515 (29) 2- - - - - SO4 ,Cl,IO3 ,CN,N3 , - - - - - ClO3 ,Br, SCN ,I, HSO4 , 2- 2- - S ,S2O3 , HSO3 - - - - - 2- The probe I ,F,Cl,Br, HCO3 ,SO4 , N/A N/A 370/420 (178) Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. 3- - - 2- PO4 ,NO3 ,NO2 ,B4O7 , 2- 2- - CO3 , HPO4 , SCN

------Probe 2 F ,Cl,Br,I,NO2 ,NO3 , 10 lM 30 min 466/523 580/663 (165) - - 3- 2- N3 , OAc ,PO4 ,CO3 , 2- 2- SO4 ,S2O3 ,H2O2, t-BuOOH, NaClO

(continued)

15 16 JIANG ET AL.

Table 6. (Continued) Reported staining Time needed Species tested for concentration before optimal Name cross-reactivity for cells signal available lex /lem (nm) References - - - - - TSSP-N3 F ,Cl,Br,I, AcO , HCO3-, 5 lM 30 min 386/460 475/590 (146) - - 3- CN-, NO2 ,NO3 ,PO4 , - - 2- 2- HS , SCN ,SO4 ,S2O3 , Cys, Hcy, GSH Lipid peroxidation probes C11-BODIPY N/A 1 lM 20 min 581/595 (40) PA derivative 1 N/A 10 lM 30 min 405/490–520 (117)

(135). Similarly, these probes respond primarily to intra- lectively and reliably measure these peroxides potentially cellular GSH under physiological conditions. Most of these due to difficulty in sensing reaction design. There are a few probes are administered to cells at low micromolar con- reported probes for lipid peroxidation, but their cross- centrations. The available common properties for glutathi- reactivity and bioavailability is not well studied (40, 117). one probes are summarized in Table 5. More work needs to be done to demonstrate their applica- bility for studying redox biology. Probes for other redox metabolites Conclusions Biochemical redox reactions involve more than just the molecules stated earlier. Continuous efforts have been Currently, the development of redox-responsive small- made to discover other important redox signaling molecules molecule fluorescent probes is primarily driven by chemists involved in cellular metabolic; some examples include in- who sometimes fail to sufficiently test their probes in com- termediates such as sulfenic acid, sulfane sulfur, sulfite, bi- plex biological systems. Although many probes with new sulfite, and peroxidized lipids. We have summarized some of reaction centers have been reported, only a few have been the recently developed probes for these metabolic interme- tested and applied by redox biologists. There is an urgent diates (Table 6). need to strengthen the communication between probe de- When thiols are biologically modified in cells, they could velopers, mostly chemists, and redox biologists. go through several intermediates such as sulfenic acid, sul- The most challenging questions for developing new redox fane sulfur, sulfite, or bisulfite. These intermediates serve probes are: (i) how to develop new sensing reactions that target different roles in cells and are important in redox biology different analytes, (ii) how to optimize these probes for real-time (57). The Carroll group has developed a series of probes quantitation in biological systems with sub-cellular resolution. targeting mostly sulfenic acid modifications on protein thiols In this contribution, we have discussed several key factors that (57–59, 83, 121, 132). These sensing reactions are mostly are important in assessing the quality of probes for both based on selective addition of sulfenic acid onto a meta- chemists and biologists, including selectivity, kinetics, and re- cyclohexanedione. They have been used for labeling of versibility of sensing reactions, quantum yield, wavelength, and sulfenic acid modified proteins from cell lysate but have Stokes shifts of fluorophores, solubility, membrane permeabil- relatively limited applications as live cell probes. ity, protein interference, turnover, and organelle specificity of The thiol derivatization agent, 4-chloro-7-nitrobenzofurazan, probe molecules. We specifically highlighted the advantages of widely used in HPLC is also used as a sulfenic acid probe (43). reversibility in sensing reactions and its application in ratio- Due to the lack of cross-reactivity tests and relatively slow metric probe design. kinetics, all the sensing reactions mentioned earlier are geared In the future, we hope that probe developers will focus not toward lysate-based measurements. only on exploring new chemistries for sensing reactions but There are also a number of sensors developed for poly- also on fine-tuning probes for biological applications. We sulfides, including sulfane sulfur initially used by the Xian believe that with further development in this field, small- group. They first utilized a biotin switch assay to distinguish molecule fluorescent probes will become useful comple- Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only. hydrodisulfides from the predominant RSH in cell lysates ments to protein-based probes in redox biology studies. (186). The same group has also developed several probes for detecting different polysulfides in live cells (20, 95, 96). Acknowledgments A few probes for sulfite and bisulfite have also been de- The research was supported in part by the National Institutes veloped based on the reductive addition of sulfite/bisulfite of Health (R01-GM115622, R01-CA207701, R21-CA213535 onto ketones or aldehydes (29, 30, 146, 165, 178). The role of to J.W., R01-AG045183, R01-AT009050, R21-EB022302, such metabolites in redox biology is not yet well established, and DP1-DK113644 to M.C.W.) and the Welch Foundation and the physiologically relevant concentration is unknown. (Q-1912 to M.C.W.). Therefore, it is currently difficult to predict which attributes are important in designing probes for sulfite and bisulfite. Author Disclosure Statement Other important redox intermediates include oxidized lipids, oxidized protein/peptide thiols, and oxidized sugar. X.J., J.C., and J.W. are co-inventors of a patent application However, there are no small-molecule probes that can se- related to the RealThiol probe. SMALL-MOLECULE REDOX FLUORESCENT PROBES 17

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Abbreviations Used (Cont.) NO$ ¼ nitric oxide 1 FRET ¼ Fo¨rster resonance energy transfer O2 ¼ singlet oxygen - GPC ¼ gel permeation chromatography O2$ ¼ superoxide - Grx ¼ glutaredoxin OCl ¼ hypochlorite GSH ¼ glutathione OH$ ¼ hydroxyl radical - GSSG ¼ oxidized glutathione ONOO ¼ peroxynitrite HClO ¼ hypochlorous acid PF1 ¼ peroxyfluor-1 H2O2 ¼ hydrogen peroxide RNS ¼ reactive nitrogen species H2S ¼ hydrogen sulfide roGFP ¼ redox-sensitive green fluorescent protein Hyper ¼ hydrogen peroxide sensor protein ROS ¼ reactive oxygen species NIR ¼ near-infrared TPP ¼ triphenlyphosphonium Downloaded by Houston Academy of Medicine Texas Medical Center Library from online.liebertpub.com at 03/02/18. For personal use only.